小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装

• Main Author: WU Fan
• Format: Article
• Language: zho
• Published: Editorial Department of Journal of Clinical Hepatology 2014-05-01
• Series: Linchuang Gandanbing Zazhi
• Subjects: RNA interference;SMAC;lentivirus
• Online Access: http://www.lcgdbzz.org/qk_content.asp?id=5855&ClassID=42414553
• ISSN: 1001-5256; 1001-5256

ObjectiveTo construct and identify the specific interference vector for second mitochondria-derived activator of caspase (SMAC) gene in mice and perform lentiviral packaging. MethodsAccording to the SMAC gene sequences in mice, three small interfering RNAs (siRNAs) (siRNA1, siRNA2, and siRNA3) and one negative control sequence (siRNAn) were designed and synthesized, and mouse hepatocytes were transfected with the above siRNAs using Lipofectamine 2000. The inhibitory effects of these siRNAs on SMAC mRNA expression were evaluated by real-time PCR, and the optimal siRNA was screened out accordingly. Oligo DNA was synthesized based on the optimal siRNA and then connected to GV115 vector to obtain recombinant interference plasmid. The candidate clones were identified by PCR and sequenced. The recombinant interference plasmid, as well as pHelper 1.0 and pHelper 2.0, was used to transfect 293T cells for lentivirus packaging. Then, cell supernatants were collected and concentrated, and the lentiviral titer was determined by gradient dilution. Comparison between groups was made by analysis of variance, and multiple comparisons were made by SNK-q test. Results The three siRNAs had different inhibitory effects on SMAC mRNA expression in hepatocytes; siRNA1 showed the strongest inhibitory effect, with an inhibition efficiency of 70.3%. Based on siRNA1, the oligo DNA was successfully synthesized and correctly connected to the lentiviral vector, as proved by sequencing. The lentiviral titer was 6×108 TU/ml, as determined by gradient dilution. ConclusionThe recombinant lentivirus that inhibits SMAC expression in mice has been constructed successfully, laying the foundation for further studies on the role of SMAC gene in liver failure.